The long-range goals of this laboratory are to analyse at both the molecular and cellular levels the mutation, evolution, transmission, segregation and recombination of mtDNA in mammalian cells and the role of mitochondrial genes in cellular energy production and intermediary metabolism. These studies relate to many areas of health science, particularly cancer biology. The principal aim during the next period of support will be to analyze the mitochondrial mutants isolated in this laboratory at the molecular level using the techniques of somatic cell genetics and DNA biochemistry. Specific projects include: a) The series of mitochondrial cytochrome b mutants will be used for sequencing studies to determine the mtDNA alterations encoding the different phenotypes. b) Wild type mutant cell lines expressing the CAP-R, PYR-IND and OLI-R mitochondrial phenotypes will be used for sequencing studies to determine whether all three phenotypes are encoded by a single or multiple mtDNA mutational events and the map positions of the sequence changes in the mitochondrial large ribosomal RNA gene. c) The mechanism of mtDNA segregation will be analyzed using both physical and genetic mtDNA markers. The genetic control of the frequencies of the PYR-IND and OLI-R phenotypes will also be analysed in a series of cybrid crosses. d) The actual mtDNA mutation frequency of an established cell line will be determined by cloning and analysing a series of mtDNA molecules.